Interaction of azimilide with neurohumoral and channel receptors.

Binding of the class III antiarrhythmic agent azimilide to brain, heart, and other organ receptors was assessed by standard radioligand binding techniques. In a survey of 60 receptors, azimilide at 10 microM inhibited binding by more than 50% at serotonin uptake (K(i): 0.6 microM), muscarinic (K(i): 0.9 to -3.0 microM), Na(+) channel site 2 (K(i): 4.3 microM), and central sigma (K(i): 6.2 microM) sites. Lesser (20-40%) inhibition was seen at adrenergic, histamine, serotonin, purinergic, angiotensin II, dopamine uptake, and norepinephrine sites and at a voltage-sensitive K(+) channel. In rat ventricle, azimilide inhibited binding to alpha(1)- and beta-adrenergic and muscarinic receptors (K(i): < 5 microM) and to the L-type Ca(2+) channel (K(i): 37.3 microM). In rat brain, azimilide blocked ligand binding to these same receptors and to a serotonin receptor, and the breadth and potency of its interaction pattern differentiated it from ten other class III antiarrhythmics. Azimilide displayed agonist and antagonist action at five muscarinic receptor subtypes in transfected NIH 3T3 cells producing receptor-sensitive mitogenesis and beta-galactosidase activity. Agonist action predominated at M(2) and M(4) subtypes, and antagonist action predominated at M(1), M(3), and M(5) subtypes. The azimilide concentration for 50% maximum stimulation (EC(50)) in M(2)-expressing cells was 1.97 microM (vs 0.14 microM for carbachol). Azimilide's receptor interactions occur at concentrations from one to forty times those required to block cardiac delayed-rectifier channels but could contribute to the efficacy and safety of the drug.

Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarization.

The neurosecretory anterior pituitary GH(4)C(1) cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K(+) reduces the dihydropyridine (+)-[(3)H]PN200-110 binding site density and (45)Ca(2+) uptake in these cells (). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca(2+) channels in GH(4)C(1) cells by membrane depolarization. Depolarization of GH(4)C(1) cells by 50 mm K(+) rapidly reduced the barium currents through L-type calcium channels by approximately 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents had an autocrine component. This study reveals a calcium-dependent down-regulation of the L-type calcium channel currents by depolarization.

Interactions of a series of fluoroquinolone antibacterial drugs with the human cardiac K+ channel HERG.

Administration of certain fluoroquinolone antibacterials has been associated with prolongation of the QT interval on the electrocardiogram and, on rare occasions, ventricular arrhythmia. Blockade of the human cardiac K+ channel HERG often underlies such clinical findings. Therefore, we examined a series of seven fluoroquinolones for their ability to interact with this channel. Using patch-clamp electrophysiology, we found that all of the drugs tested inhibited HERG channel currents, but with widely differing potencies. Sparfloxacin was the most potent compound, displaying an IC50 value of 18 microM, whereas ofloxacin was the least potent compound, with an IC50 value of 1420 microM. Other IC50 values were as follows: grepafloxacin, 50 microM; moxifloxacin, 129 microM; gatifloxacin, 130 microM; levofloxacin, 915 microM; and ciprofloxacin, 966 microM. Block of HERG by sparfloxacin displayed a positive voltage dependence. In contrast to HERG, the KvLQT1/minK K+ channel was not a target for block by the fluoroquinolones. These results provide a mechanism for the QT prolongation observed clinically with administration of sparfloxacin and certain other fluoroquinolones because free plasma levels of these drugs after therapeutic doses approximate those concentrations that inhibit HERG channel current. In the cases of levofloxacin, ciprofloxacin, and ofloxacin, inhibition of HERG occurs at concentrations much greater than those observed clinically. The data indicate that clinically relevant HERG channel inhibition is not a class effect of the fluoroquinolone antibacterials but is highly dependent upon specific substitutions within this series of compounds. HERG channel affinity should be an important criterion for the development of newer fluoroquinolones.

Permanently charged chiral 1,4-dihydropyridines: molecular probes of L-type calcium channels. Synthesis and pharmacological characterization of methyl(omega-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodide, calcium channel antagonists.

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydropyridines and methyl (omega)-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and (S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-+ ++pyridinecarboxylic acid, obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridi necarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-110 and the block of whole cell barium currents through L-type calcium channels in GH4C1 cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.

Pharmacological receptors: a century of discovery--and more.

A brief survey of the history of the development of the concept of the pharmacological receptor is presented. From the pioneering concepts of Paul Ehrlich, John Langley and others, receptors are described in terms of their recognition properties, their structures, transducing abilities and the impact of genomics and their role in contributing to genetic diseases. The receptor concept has firmly underpinned our advances in drug development and molecular medicine of the latter half of this century and it is clear that it will continue to drive pharmaceutical developments in the 21st century.

Hijacked receptors.

Pharmacological receptors are typically defined by their selectivity of ligand recognition, including where appropriate stereoselectivity of interaction. It is increasingly clear that receptors may, in fact, be promiscuous species. This promiscuity arises at several levels of organization: two appear to be of particular importance. A given ligand-receptor complex may couple with different effectors and may generate quite different physiological responses: this is particularly common, although not uniquely so, for G protein-coupled receptors. Or a single receptor may recognize fundamentally different ligands often of significantly different characteristics: a number of viruses gain entry to cells through their interaction at receptors for neurotransmitters, peptides or hormones.

Lipophilic 4-isoxazolyl-1,4-dihydropyridines: synthesis and structure-activity relationships.

A series of 4-isoxazolyl-1,4-dihydropyridines bearing lipophilic side chains at the C-5 position of the isoxazole ring have been prepared. The calcium channel antagonistic activity of these compounds has been evaluated. A hypothetical model for binding of these compounds in the calcium channel is proposed, and the validity of this model is evaluated based on the SAR of this series of calcium binding, especially for the two most active derivatives, 1a, g. The solid-state structure for the most active compound, 1a, has also been determined, and its important features are reported.

The pharmacology of ion channels: with particular reference to voltage-gated Ca2+ channels.

Ion channels are molecular machines that serve as principal integrating and regulatory devices for the control of cellular excitability. They are also major targets for drug action. The basic aspects of ion channel structure and pharmacological control are reviewed and illustrated with specific reference to a major class of therapeutic agents and molecular tools--the clinically available Ca2+ channel antagonists.

Rho Chi lecture. Pharmaceutical sciences in the next millennium.

Even a cursory survey of this article suggests that the pharmaceutical sciences are being rapidly transformed under the influence of both the new technologies and sciences and the economic imperatives. Of particular importance are scientific and technological advances that may greatly accelerate the critical process of discovery. The possibility of a drug discovery process built around the principles of directed diversity, self-reproduction, evolution, and self-targeting suggests a new paradigm of lead discovery, one based quite directly on the paradigms of molecular biology. Coupled with the principles of nanotechnology, we may contemplate miniature molecular machines containing directed drug factories, circulating the body and capable of self-targeting against defective cells and pathways -- the ultimate "drug delivery machine." However, science and technology are not the only factors that will transform the pharmaceutical sciences in the next century. The necessary reductions in the costs of drug discovery brought about by the rapidly increasing costs of the current drug discovery paradigms means that efforts to decrease the discovery phase and to make drug development part of drug discovery will become increasingly important. This is likely to involve increasing numbers of "alliances," as well as the creation of pharmaceutical research cells -- highly mobile and entrepreneurial groups within or outside of a pharmaceutical company that are formed to carry out specific discovery processes. Some of these will be in the biotechnology industry, but an increasing number will be in universities. The linear process from basic science to applied technology that has been the Western model since Vannevar Bush's Science: The Endless Frontier has probably never been particularly linear and, in any event, is likely to be rapidly supplanted by models where science, scientific development, and technology are more intimately linked. The pharmaceutical sciences have always been an example of use-directed basic research, but the relationships between the pharmaceutical industry, small and large, and the universities seems likely to become increasingly developed in the next century. This may serve as a significant catalyst for the continued transformation of universities into the "knowledge factories" of the 21st century. Regardless, we may expect to see major changes in the research organizational structure in the pharmaceutical sciences even as pharmaceutical companies enjoy record prosperity. And this is in anticipation of tough times to come.

The physiological and pharmacological significance of cardiovascular T-type, voltage-gated calcium channels.

An influx of calcium ions into cells, made possible by the opening of specific, voltage-gated channels, triggers muscular contraction and several other physiological processes. Two types of calcium channels, L-type and T-type, are found in the cardiovascular system. These two types of channels differ considerably in their electrical and chemical characteristics and in their distribution in tissue. The L-type calcium channel is responsible for normal myocardial contractility and for vascular smooth muscle contractility. In contrast, T-type calcium channels are not normally present in the adult myocardium, but are prominent in conducting and pacemaking cells. They are thought to help regulate vascular tone, signal conduction, cardiac pacemaking, and the secretion of certain intercellular transmitters. T-Type channels also seem to have an important role in normal growth processes and in the tissue remodeling that occurs in pathologic processes such as cardiac hypertrophy. Traditional calcium antagonists act on L-type channels. Mibefradil is a recently characterized calcium antagonist and the first that is selective for T-type calcium channels. This unique property may lead to major applications in cardiovascular medicine.

Cardiovascular T-type calcium channels: physiological and pharmacological significance.

CELLULAR CALCIUM REGULATION: A variety of Ca2+ control processes are responsible for Ca2+ homeostasis and signaling. Voltage-gated Ca2+ channels are dominant in the cardiovascular system. VOLTAGE-GATED Ca2+ CHANNELS: There are several distinct subclasses of Ca2+ channels, distinguished by location, biophysical, structural and pharmacological characteristics. They include both high- and low-voltage-activated channels. The long-lasting (L) type of high-voltage-activated channel is well characterized and is the site of action for the existing clinically available Ca2+ channel antagonists: nifedipine, verapamil and diltiazem. T-TYPE Ca2+ CHANNELS: The low-voltage-activated transient (T-type) channel is widespread in the cardiovascular system and in neurons. It serves pacemaking functions and supports Ca2+ signaling in secretory cells and vascular smooth muscle. The T-type channel also functions in cell growth processes under physiological and pathological conditions. MIBEFRADIL AS A T-TYPE Ca2+ CHANNEL ANTAGONIST: Mibefradil (Ro 40-5967) is a structurally novel Ca2+ antagonist with selectivity for T-type over L-type channels. This selectivity may underlie its vasodilating activity and heart rate depressive effect, its lack of negative inotropy and its cardioprotective properties.

Pharmacologic and therapeutic differences among calcium channel antagonists: profile of mibefradil, a new calcium antagonist.

Calcium antagonists are a heterogeneous group of drugs, each with a different chemical structure and cardiovascular profile. Distinguishing factors include pharmacokinetics, mode of calcium mobilization affected, class and subclass of calcium channel inhibited, state-dependent interactions, and effect of disease on the drug's activity. A new calcium antagonist, mibefradil, has a unique chemical structure and cardiovascular profile compared with those currently available, and it appears to represent a new class of calcium antagonists.

Depolarization as a regulatory signal at voltage-gated calcium channels.

Changes in membrane potential are a regulatory signal for voltage-gated ion channels including the family of Ca2+ channels. This regulatory role includes the voltage-dependent opening, closing, and inactivation of ion channels and the control of drug access and affinity for discrete channels states. Membrane potential is both a short- and long-term regulatory signal controlling the number and function of these voltage-gated channels. Depolarization of neuronal and neurosecretory cells produces down-regulation of L-type voltage-gated Ca2+ channels. These processes are described and their relevance to physiologic and pathologic processes of neuronal development and neuroprotection are indicated.

Ion channels as pharmacologic receptors: the chirality of drug interactions.

Ion channels are pharmacologic receptors and as such exhibit stereoselective interactions with drugs. Ion channels are conformationally mobile transmembrane proteins existing in a number of open and closed states. Drug interactions with these different states may differ quantitatively and qualitatively. Stereoselectivity may not be a constant factor and may change according to channel state as determined by stimulus mode or experimental conditions. Selected examples are cited for Na+ and Ca2+ channels.

Structure-function relationships of calcium antagonists. Effect on oxidative modification of low density lipoprotein.

Human low density lipoprotein (LDL) incubated with active Ca2+ antagonists from three different chemical groups, 1,4-dihydropyridines that are of reduced activity as Ca2+ antagonists, vitamin E, and probucol, was more resistant than control to copper- or human monocyte-induced oxidation, as assessed by thiobarbituric acid reactive substance (TBARS) content, degradation by J774 macrophages, and relative electrophoretic mobility on agarose gel. In the copper-induced oxidation system, the drugs tested reduced the TBARS levels of LDL in a concentration-dependent manner. The order of potency was vitamin E > felodipine > 2-chloro analog of nifedipine > nifedipine > amlodipine, nitrendipine, verapamil > diltiazem. In agreement with the results of the TBARS assay, felodipine (25 microM) was also the most effective calcium antagonist in the degradation assay, inducing a significant (P<0.05) 97 +/- 2% reduction in the amount of oxidized [125I]LDL degraded by J774 macrophages compared with nifedipine and its 4-nitro analog, amlodipine, and verapamil. The relative mobility of oxidized LDL on agarose gel was reduced significantly (P<0.05) by felodipine (50 microM) and amlodipine (25 and 50 microM) when compared with control, and was similar to that of native LDL, suggesting an effect of these drugs on the net negative charge of oxidized LDL. In the cell-induced oxidation system, both nifedipine and felodipine (25 microM) induced significant (P<0.05) reductions in the TBARS content of LDL (96 +/- 2 and 65 +/- 9%, respectively) compared with amlodipine, verapamil and the 4-nitro analog of nifedipine. However, in this oxidation system nifedipine was a more effective antioxidant than felodipine. Analysis of the structure-function relationships for the effect of 1,4-dihydropyridines on the oxidative modification of LDL suggests an important role for the 2-substitution of the phenyl ring, and an essential role for the dihydropyridine ring. This study clearly shows that Ca2+ antagonists from different chemical groups have a concentration-dependent effect as antioxidants against LDL oxidation. However, the order of potency of the drug depends on the oxidation system and the assay used to measure the antioxidant effect. Our data suggest that such a protective effect of Ca2+ antagonists against LDL oxidation could play a role in the antiatherosclerotic effect of these drugs.